The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
The biological revolution has progressed from genomics to proteomics as the newest frontier for gathering information concerning cellular physiology. Transcription of DNA is an early step in an extended process resulting ultimately in the expression of genomic information as a functional protein. Additional steps can include processing of the initial transcript to mRNA, translation of the mRNA into protein, and posttranslational processing of the protein (e.g., cleavage of the protein into smaller fragments, modification of the protein by glycosylation, methylation, acylation, phosphorylation, etc.). In addition, the protein may be activated or deactivated by interaction with other proteins and/or with small molecules (e.g., cofactors). Regulation of active protein expression can occur at one or more of these steps, and the amount of active protein in the cell at any time will vary widely with the state of the protein in the cell. Thus, the presence of a given gene in a cell's genome, or the total amount of a particular protein in a cell is not necessarily a good prognosticator of the state of the cell, as reflected by the amount of active protein present in the cell.
In evaluating candidate drugs, the readout should provide an indication how the drug will perform in vivo. For example, an accurate evaluation of a candidate drug can be obtained by using the drug in vivo and determining the effect of the drug on the indication and/or absorption, distribution, metabolism, and excretion (“ADME”) studies performed. However, where there are a large number of candidates as are available today from combinatorial libraries, and natural and other sources, substitute procedures must be available to allow for reducing the number of candidate drugs to be studied. To allow for large numbers of candidate drugs to be evaluated, while having reasonable costs and times involved, cellular surrogates are finding use. One can expose cells in culture to candidate drugs. The question then is what should be analyzed to obtain the greatest amount of accurate information relevant to the effect of the drug in the most expedient way.
There is substantial interest in providing platforms that will provide answers to questions asked about the effect of candidate drugs on cells, tissues, and/or organisms. In order for platforms to be useful they should be efficient, reliable, and economic, and maximize the information provided and the predictive capability of the results. The ability to analyze samples in parallel and the reproducibility, speed, automation, sensitivity, and specificity of the analysis procedures can all contribute to maximizing the efficiency and reliability of such a platform.
Numerous methods have been described for analyzing protein compositions. Some typical examples include WO 00/11208, which discusses mass spectrometric methods for analysis of proteins; Cravatt and Sorenson, Current Opinion in Chemical Biology (2000) 4(6): 663–668, which discusses chemical strategies for analyzing protein function; U.S. Pat. No. 4,433,051, which discusses the use of α-difluoromethylomithine for use in protein analysis; U.S. Pat. No. 6,127,134, which discusses difference gel electrophoresis using matched multiple dyes; Gygi et al., Proc. Natl. Acad. Sci. USA (2000) 97:9390–5, which discusses the use of 2D gel electrophoresis in conjunction with mass spectrometry to analyze yeast proteins; and Aebersold et al., PCT/US99/19415, which discusses digestion of labeled protein samples.
Complex protein mixtures, such as proteomes, can be difficult to analyze. Not only are there many components in the mixtures, but as samples of these mixtures may be processed, many artifacts can be introduced into the sample, e.g., by hydrolysis of amide bonds, deamination, oxidation and the like. In addition, proteins present in the mixture that originally derived from the same polypeptide sequence may have been subject to differential processing reactions, such as glycosylations, prenylations, etc. Moreover, in analysis procedures in which the proteins in a complex mixture are subject to proteolysis, the total number of components is greatly increased in comparison to the original sample. As a result, numerous fractions in a chromatography procedure or bands in an electrophoretic gel can be related to a single protein in the original sample, greatly complicating the interpretation of the data. It is of interest to find ways to simplify the compositions that are being analyzed to permit a more accurate and robust interpretation of the observed results.